Traditional cancer therapies, such as surgery, radiation, and chemotherapy, are often insufficient in treating patients and usually cause severe side effects.
Immunotherapy has shown promise as an alternative treatment method with less negative side effects.
It is now well established that the immune system has cells, particularly CD8+ cytotoxic T lymphocytes (CTLs), which can recognize and potentially kill tumor cells. Nevertheless, a major problem is that the killing ability of these T cells are either not induced or only weakly induced in cancer patients. One possibility is that there is inadequate tumor antigen presentation and co-stimulation by dendritic cells (DCs), “nature's adjuvant” for eliciting a functional and tumor-specific T cell immunity in cancer patients.
Existing cancer immunotherapy strategies mainly focus on antigen-loaded autologous, patient-derived DCs, which have been differentiated and antigen-loaded ex vivo. The underlying premise of this approach is that the efficiency and control provided by ex vivo manipulation of the DCs generates DCs with strong antigen-presenting and co-stimulatory capacity. The quality of the T cell response depends on the ability of these autologous DCs to present tumor antigens to T cells in a MHC-restricted manner (DCs and T cells have to be from the same individual) in draining lymph nodes and thus create a tumor-specific T cell response.
Monocyte-derived, autologous DCs are the most commonly used DCs in pilot studies, as it is possible to obtain billions of monocytes from peripheral blood leukocytes collected by leukapheresis, a laborious and time-consuming procedure in which white blood cells are separated from circulating blood. Several methods are available to subsequently enrich monocytes and two of these methods, elutriation and antibody/bead isolation, can also be performed in conformity with Good Manufacturing Practice (GMP) guidelines.
The monocytes are subsequently cultivated in media supplemented with GM-CSF and IL-4 for 4-7 days, leading to their differentiation into immature DCs, which immature DCs are characterized by their outstanding capacity to produce large amounts of pro-inflammatory chemokines and cytokines upon subsequent stimulation with certain types of activating factors (Sallusto et al, Eur J Immunol, 1999. 29:1617; Napolitani et al, Natuer Immunology 2005. 6:769). The stimulated DCs are usually pre-pulsed with relevant tumor antigen(s) and activated for 1-2 days before vaccination. However, the immune responses to such DC-based vaccines are often weak, and clinical responses are rarely complete and long lasting.
Little has been known regarding the fate and function of ex vivo generated autologous DCs after they have been injected. In the human setting, the migration pattern of injected vaccine DCs was recently tracked in vivo and notably, less than 5% of the injected DCs reached the draining lymph nodes while the majority of DCs remained at the injection site. These locally trapped vaccine DCs rapidly lost their viability and were subsequently cleared by recruited antigen-presenting cells.
Data has now been provided that injected vaccine DCs that have been activated ex vivo during a limited time-period (i.e. 6 to 18 h) become pro-inflammatory (PI) DCs, which are able to indirectly prime native CD8+ T cells in vivo by acting as a pure local immune adjuvant. This adjuvant function of injected PI-DCs is strictly dependent on their ongoing secretion of certain DC and NK-cell recruiting chemokines at the time of administration (after removal of activating factors). Such PI-DCs also express/secrete factors that induce activation of recruited endogenous NK-cells and DCs at the vaccination site. In contrast to PI-DCs, long-time (i.e. >24 h) activated DCs, which have been commonly used in clinical trial, are characterized by their “exhausted” state (Langenkamp et al 2000), and therefore unable to secrete desirable chemokines and DC-activating factors at the time of administration.
In conclusion, PI-DCs not only can act as direct stimulators of MHC-compatible autologous T cells but also act as an adjuvant producing large quantities of pro-inflammatory chemokines and cytokines at the time of administration. Local injection of PI-DCs will lead to recruitment and activation of other immune cells, including circulating NK cells and DC-precursors. If the injected PI-DCs have been preloaded with relevant tumor antigens or injected directly into an existing tumor lesion, recruited endogenous DCs will engulf dying vaccine cells expressing relevant tumor antigens or dying antigen-expressing tumor cells, respectively. After activation these recruited and subsequently antigen-loaded endogenous DCs will migrate to draining lymph nodes were they prime tumor-specific T cells in a MHC-restricted manner (Liu et al, 2008). This conclusion is supported by data from several recent pre-clinical studies in which tumor growth was significantly reduced by therapeutic vaccinations with non-exhausted MHC-incompatible, allogeneic PI-DCs (Alder et al 2008, Siders et al 2009, Edlich et al 2010)
The strong adjuvant function by PI-DCs, which importantly don't require MHC-compatibility between PI-DCs and patient T cells, therefore introduces the possibility of using pre-produced and freeze-stored MHC-incompatible, allogeneic, PI-DCs as “off the shelf” vaccines, representing a viable, practical alternative to the current custom-made, patient-specific DC vaccines. The use of such MHC-incompatible, allogeneic, PI-DCs is disclosed in EP 1 509 244 B1 and WO 2011/098516.
For ethical reasons, large scale procurement of monocytes from normal blood donors by leukapheresis for the sole purpose of commercial large-scale vaccine production for clinical use is not feasible. In practice, the available raw material, i.e. monocytes, for PI-DC production is therefore restricted to monocytes obtained from waste-product (buffy coats and/or used leukocyte depeltion filteers) in the course of separating unwanted leukocytes from different whole blood components or monocytes obtained from buffy coats at blood banks.
However, the total number of monocytes which can be isolated from each buffy coat or from each blood bag-leukocyte depletion filter is usually less than 200 millions (Ebner S et al., Generation of large numbers of human dendritic cells from whole blood passaged through leukocyte removal filters: an alternative to standard buffy coats J Immunol Methods 252 (2001), leading to unacceptable high costs for enrichment and subsequent DC-differentiation of separate batches of monocytes (each batch derived from one single donor) with methods that are in conformity with Good Manufacturing Practice (GMP) guidelines according to the art.
Thus, there is a need in the art for a method for large scale and cost-effective clinical grade production of non-exhausted immature dendritic cells from monocyte-containing waste-product derived from blood banks.